Olga A. Pavlova

2-[18F]F-A-85380: PET imaging of brain nicotinic acetylcholine receptors and whole body distribution in humans

Noninvasive imaging of nicotinic acetylcholine receptors (nAChRs) in the human brain in vivo is critical for elucidating the role of these receptors in normal brain function and in the pathogenesis of brain disorders. Here we report the first in vivo visualization of human brain areas containing nAChRs by using PET and 2‐[18F]fluoro‐3‐(2(S)azetidinylmethoxy)pyridine (2‐[18F]FA). We acquired scans from six healthy non‐smoking volunteers after i.v. bolus administration of 2‐[18F]FA (1.6 MBq/kg or 0.043 ± 0.002 mCi/kg). This dose was sufficient for visualizing nAChRs in the thalamus up to 5 h after injection. There were no adverse effects associated with administration of no‐carrier‐added 2‐[18F]FA (1.3‐10 pmol/kg). Consistent with the distribution of nAChRs in human brain, accumulated radioactivity was greatest in thalamus, intermediate in the midbrain, pons, cerebellum, and cortex; and least in white matter. As ˜90% of the injected radioactivity was eliminated via the urine (biological half‐life ca. 4 h), the urinary bladder wall received the highest radiation dose. The estimate of radiation dose equivalent to the urinary bladder wall (ca. 180 ± 30 mSv/MBq or 0.7 rem/mCi with a 2.4 h void interval) suggests that multiple studies could be performed in a single subject. The results predict that quantitative PET imaging of nAChRs in human brain with 2‐[18F]FA is feasible.

Greater Nicotinic Acetylcholine Receptor Density in Smokers Than in Nonsmokers: A PET Study with 2-18F-FA-85380

Assays of human postmortem brain tissue have revealed that smokers have greater densities of high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain regions than do nonsmokers or exsmokers. Quantitative PET imaging of nAChRs in humans has recently been reported using the α4β2* subtype–specific radioligand 2-18F-FA-85380 (2FA). Methods: We used PET and 2FA to measure total volumes of distribution corrected for the free fraction of 2FA in plasma (VT/fP) in 10 nonsmokers and 6 heavy smokers (>14 cigarettes/d; abstinent for >36 h). Dynamic PET scans were performed over 8 h, commencing immediately after a bolus injection of 2FA. Anatomic sampling was performed on PET images that were coregistered to MR images acquired from each volunteer. Data were analyzed by Logan plots and by 1- and 2-tissue-compartment models using unbound, unmetabolized arterial 2FA concentration as the input function. Results: All modeling methods yielded similar results. VT/fP was significantly higher in smokers than in nonsmokers in all brain regions tested, except the thalamus. We used measures of VT/fP and estimates of nondisplaceable volume of distribution and found 25%–200% higher values in smokers than in nonsmokers for the volume of distribution for the specific binding compartment in the frontal cortex, midbrain, putamen, pons, cerebellum, and corpus callosum. These findings were consistent with voxel-based analysis using statistical parametric mapping. Conclusion: Our findings suggest that PET with 2FA can be used to study the role of nicotine-induced upregulation of nAChRs in active smokers and during smoking cessation.

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

Significance Most proteins that reside in the bacterial outer membrane are β sheets that fold into a unique cylindrical structure known as a “β barrel.” Here we describe significant insights into the function of the Bam complex, a protein machine that catalyzes the insertion of β barrel proteins into the membrane by an unknown mechanism. By analyzing the assembly of autotransporters, a specialized family of outer membrane proteins, we found that the function of the Bam complex can be divided into an initial substrate binding stage and a subsequent insertion stage that is surprisingly sensitive to structural distortions in client proteins. Autotransporters are bacterial virulence factors that contain an N-terminal extracellular (“passenger”) domain and a C-terminal β barrel (“β”) domain that anchors the protein to the outer membrane. The β domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the β domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the β domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the β domain into the outer membrane, but do not delay the binding of the β domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the β domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of β barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.

Evaluation of 5‐(2‐(4‐pyridinyl)vinyl)‐6‐chloro‐3‐(1‐methyl‐2‐(S)‐pyrrolidinylmethoxy)pyridine and its analogues as PET radioligands for imaging nicotinic acetylcholine receptors

A novel series of compounds derived from the high‐affinity nicotinic acetylcholine receptor (nAChR) ligand, 5‐(2‐(4‐pyridinyl)vinyl)‐6‐chloro‐3‐((1‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)pyridine (Me‐p‐PVC), originally developed by Abbott Laboratories, was characterized in vitro in nAChR binding assays at 37°C to show Ki values in the range of 9–611 pm. Several compounds of this series were radiolabeled with 11C and evaluated in vivo in mice and monkeys as potential candidates for PET imaging of nAChRs. [11C]Me‐p‐PVC (Ki =56 pm at 37°C; logD = 1.6) was identified as a radioligand suitable for the in vivo imaging of the α4β2* nAChR subtype. Compared with 2‐[18F]FA, a PET radioligand that has been successfully used in humans and is characterized by a slow kinetic of brain distribution, [11C]Me‐p‐PVC is more lipophilic. As a result, [11C]Me‐p‐PVC accumulated in the brain more rapidly than 2‐[18F]FA. Pharmacological evaluation of Me‐p‐PVC in mice demonstrated that the toxicity of this compound was comparable with or lower than that of 2‐FA. Taken together, these results suggest that [11C]Me‐p‐PVC is a promising PET radioligand for studying nAChR occupancy by endogenous and exogenous ligands in the brain in vivo.

Radiosynthesis of 5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-[11C]methyl-2-(S)-pyrrolidinylmethoxy)pyridine, a high affinity ligand for studying nicotinic acetylcholine receptors by positron emission tomography

5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine (1b) exhibited high affinity for nicotinic acetylcholine receptors in the in vitro competition binding assays, with a K(d) value in the low picomolar range, performed at room temperature and at physiological temperature. An efficient radiochemical synthesis of 5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-[(11)C]methyl-2-(S)-pyrrolidinylmethoxy)pyridine (1c), a potential tracer for the study of nAChR by positron emission tomography, has been developed.

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6‐chloro‐3‐((2‐(S)‐azetidinyl)methoxy)‐5‐(2‐fluoropyridin‐4‐yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The Kd and T1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2‐[18F]fluoro‐3‐(2(S)‐azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α4β2* nAChRs distribution. Comparison between [18F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [18F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non‐displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [18F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α4β2* versus α3β4* nAChRs. These results suggest that [18F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.